What are the Differences Between DNA and RNA Probes?

In the last decade, tremendous progress has been made in the field of molecular diagnostics. Many new nucleic acid-based diagnostic tools or assays have been developed that allow analysis of DNA and RNA molecules in clinical samples. These assays are now routinely used for monitoring or detecting, as well as to help decide which therapies would work best for patients. Specific molecular probes and primers are designed for this purpose. Gene probes are used in various blotting and in situ hybridization (ISH) techniques for the detection of nucleic acid sequences in food industry, environmental, medical and veterinary applications to improve the specificity of the analyses. In medicine, they can help in the identification of microorganisms and the diagnosis of infectious, inherited, and other diseases. In practice, double and single standard DNAs, mRNAs, and other RNAs synthesized in vitro are all used as probes. DNA - RNA probe assays are faster and sensitive so that many conventional diagnostic tests for viruses and bacteria involving culturing of the organisms are being fast replaced by molecular probe assays. While culture tests can take days, molecular probe assays can be performed within a few hours or minutes. Molecular probes can be broadly categorized into DNA probes and RNA probes, cDNA probes and synthetic oligonucleotide probes can also be used for various purposes. At Enzo, we offer a complete set of tools for nucleic acid labeling and detection.

Nucleic acid probes are either a single stranded DNA or an RNA with a strong affinity towards a specific DNA or RNA target sequence. This affinity and complementary sequence allows binding to specific regions of a target sequence of nucleotides. The degree of homology between target and probe results in stable hybridization. In developing a probe, a sequence of nucleotides must be identified, isolated, reproduced in sufficient quantity, and tagged with a label that can be detected. In theory, any nucleic acid can be used as a probe provided it can be labeled to permit identification and quantitation of the hybrid molecules formed between the probe and sequence to be identified.

Choice of Label

Probes can be labeled either by radioactive isotopes or can also be labeled with nonradioactive molecules such as biotin, digoxegenin etc. However, the use of radioisotope labeled probes is limited by the short half-life of the isotope, and economic and environmental aspects of radioactive waste disposal.

Advances in nucleic acid technology offer alternatives to radioactively labeled probes. The use of nonradioactive labels have several advantages such as safety and higher efficiency of the labeling reaction. One example is biotin labeling of nucleic acids. This system exploits the affinity that the glycoprotein avidin has for biotin. These probes can be prepared in advance in bulk and stored at -20°C for repeated uses. Digoxigenin is another chemical derived from plants and used for non-radioactive labeling of probes. An antibody associated with an enzyme (antidigoxigenin - alkaline phosphatase conjugate) is used for the detection of the presence of digoxigenin.

Progress in sequence-specific DNA imaging by fluorescence microscopy has been achieved by employing the fluorescent hybridization in situ (FISH) method. This type of label is especially useful for the direct examination of microbiological or cytological specimens under the microscope.

Figure 1: Fluorescence emission profiles of available fluorescent labeled dUTPs. The dye-dUTPs are designed to perform especially well in multi-color applications, such as in situ hybridization and microarray analysis.

What are DNA probes?

A DNA probe is a fragment of DNA that contains a nucleotide sequence specific for the gene or chromosomal region of interest. DNA probes employ nucleic acid hybridization with specifically labeled sequences to rapidly detect complementary sequences in the test sample. A variety of methodologies for labeling DNA have been described. In short, these methods are used to generate end-labeled or continuously labeled probes. Most enzyme-mediated labeling techniques are very much dependent on polymerase activity, which is responsible for incorporation of the labeled nucleotides. Furthermore, the use of Taq or other thermostable DNA polymerases permits labeling reactions to be performed at higher temperatures via PCR, thereby reducing the incidence of enzyme-mediated point mutations during probe synthesis. PCR is an excellent method for probe synthesis, requiring very small quantities of template material. In the presence of appropriately labeled nucleotide primers, PCR products are labeled as they are being synthesized. Alternatively, the primers themselves may be labeled non-isotopically during their own synthesis, negating the requirement for the inclusion of labeled nucleotide precursors as part of the reaction mix. Random priming is a type of primer extension in which a mixture of small oligonucleotide sequences, acting as primers, anneal to a heat-denatured double-stranded template. The annealed primers ultimately become part of the probe itself, because the Klenow fragment of DNA polymerase I extends the primers in the 3′ direction and, in so doing, incorporates the label. Nick translation is one of the oldest probe labeling techniques. It involves randomly nicking the backbone of a double-stranded DNA with dilute concentrations of DNase I. At extremely low concentrations, this enzyme nicks a template at four or five sites, producing a free 3′-OH group that can act as a primer at each nicking location. Next, the enzyme DNA polymerase I removes the native nucleotides from the probe molecules in the 5′→3′ direction (exonuclease activity) while replacing them with labeled dNTP precursors by virtue of its 5′→3′ polymerase activity. Nick translation is efficient for both linear and covalently closed DNA molecules, and labeling reaction are completed in less than an hour.

Enzo offers a Nick Translation DNA Labeling System 2.0 to provide a simple and efficient method for generating labeled DNA. The kit can accommodate a wide range of fluorophore-labeled, biotin-labeled, and digoxigenin-labeled nucleotides. In addition to choice of label, the kit design allows the user to optimize incorporation and product size by adjusting the ratio of labeled-dUTP to dTTP. The ready-to-use NT Enzyme Mix is user friendly and minimizes error from pipetting. Probes labeled by nick translation can be used in many different hybridization techniques including: in situ hybridization (ISH), fluorescent in situ hybridization (FISH), screening gene banks by colony or plaque hybridization, DNA or RNA transfer hybridization, and re-association kinetic studies.

What are RNA probes?

RNA probes are stretches of single-stranded RNA used to detect the presence of complementary nucleic acid sequences (target sequences) by hybridization. RNA probes are usually labeled, for example with radioisotopes, epitopes, biotin or fluorophores to enable their detection. RNA probes as hybridization tools remain popular because of several key advantages associated with their use. These probes are synthesized by in vitro transcription and can be substituted for DNA probes in nearly all applications. High specific activity RNA probes or riboprobes may also be synthesized from DNA templates cloned in expression vectors such as SP 6 and T 7 systems. RNA probes are single-stranded and offer several advantages over DNA probes including improved signal or hybridization blots. Compared to the diverse methods for DNA probe synthesis, there is only one reliable method for labeling RNA probes, namely in vitro transcription. Because of the intrinsically labile nature of RNA and the susceptibility to RNase degradation, RNA probes must be treated with the same care as any other RNA preparations.

in vitro transcription is a reliable and economical method for generating RNA probes. Large amounts of efficiently labeled probes of uniform length can be generated by transcription of a DNA sequence ligated next to an RNA promoter. One excellent strategy is to clone the DNA to be transcribed between two promoters in opposite orientations. This allows either strand of the cloned DNA sequence to be transcribed in order to generate sense and antisense RNA for hybridization studies. One alternative method to generating continuously labeled RNA probes by in vitro transcription is to label the 5′ end of the molecule. This method of 5′ end-labeling is colloquially known as the kinasing reaction; it specifically involves the transfer of the γ phosphate of ATP to a 5′-OH substrate of RNA or DNA (forward reaction). The forward kinasing reaction is far more efficient than the exchange reaction which involves the substitution of 5′ phosphates.

Probe synthesis by 3′ end-labeling involves the addition of nucleotides to the 3′ end of either DNA. DNA 3′ end-labeling is most often catalyzed by terminal transferase. Single- and double-stranded DNA molecules are labeled by the addition of dNTP to 3′-OH termini. RNA can also be 3′ end-labeled using the enzyme poly(A) polymerase. This enzyme, which is naturally responsible for nuclear polyadenylation of many heteronuclear RNAs, catalyzes the incorporation of Adenosine Mono Phosphate. Isotopic labeling requires α-labeled ATP precursors. In addition to its utility in RNA probe synthesis reactions, poly(A) polymerase can be used to polyadenylate naturally poly(A)– mRNA and other RNAs in order to support oligo(dT) primer-mediated synthesis of cDNA.

Use of Probes in Research Applications

In Northern blotting, the RNA under study is fractionated by gel electrophoresis. The molecules are then transferred to a membrane that is incubated with the labeled probe(s). Hybridization of complementary sequences allows visualization of target RNA sequence. Southern blotting involves the fractionation and transfer of DNA to membranes. Membranes are then incubated with the labeled DNA probe(s). Hybridization of complementary sequences allows visualization of target DNA sequence. Other applications involving ISH and FISH experiments allow for the localization of RNA or DNA targets in cells and tissues. This technique uses cultured cells or tissue section samples for hybridization and detection of the gene or target sequence of interest. Cells or tissues are processed so that their endogenous nucleic acids are fixed in place, but available for hybridization to and detection by labeled probes.

Figure 2: Enzo Life Sciences offers a complete set of solutions for in situ hybridization, providing everything you need for labeling, hybridization, and detection.

Advances in single-cell analysis technologies are providing novel insights into phenotypic and functional heterogeneity within seemingly identical cell populations. Techniques for profiling and understanding RNA expression at single-cell resolution have rapidly progressed in recent years.
Enzo Life Sciences is a recognized global leader in providing DNA and RNA labeling technologies with several key patents in developing biotin and fluorescent labeled nucleotide probe for gene expression studies. We offer a range of products for Genomics research needs. For a simple and efficient method for generating labeled DNA, please check out our Nick translation DNA labeling kit as well as a list of our SEEBRIGHT® fluorescent dye-dUTPs and our Allylamine-dUTP. For all questions and concerns regarding any of our products, our Technical Support Team is here to assist.